Subtractive transgenics

Abstract

Described herein are methods for predictable, highly selective expression of a transgene in a human or non-human animal. The methods comprise subtractive transgenics, wherein the expression pattern of one selective promoter is subtracted from the expression pattern of a second selective promoter. Provided are non-human transgenic animals exhibiting a highly selective expression pattern and methods of producing such animals. Further provided are methods of selective expression of a transgene in an animal, including a human, by administration of viral vectors.

1 . A transgenic non-human animal comprising:
 (a) a first heterologous nucleic acid encoding a recombinase operably linked to a first selective promoter;   (b) a second heterologous nucleic acid encoding a transcription factor operably linked to a second selective promoter, wherein expression of the transcription factor is regulated by expression of the recombinase; and   (c) a third heterologous nucleic acid comprising a transgene under the transcriptional control of a response element specific for the transcription factor.   
     
     
         2 . The transgenic non-human animal of  claim 1 , wherein the recombinase is expressed in a first population of cells and the transcription factor is expressed in a second population of cells, wherein the first and second populations of cells comprise at least one overlapping sub-population of cells and at least one non-overlapping sub-population of cells. 
     
     
         3 . The transgenic non-human animal of  claim 2 , wherein the transgene is expressed only in the at least one overlapping sub-population of cells. 
     
     
         4 . The transgenic non-human animal of  claim 2 , wherein the transgene is expressed only in the at least one non-overlapping sub-population of cells. 
     
     
         5 . The transgenic non-human animal of  claim 1 , wherein expression of the transcription factor is disabled by expression of the recombinase. 
     
     
         6 . The transgenic non-human animal of  claim 5 , wherein the second heterologous nucleic acid comprises recombinase recognition sites flanking the transcription factor, such that expression of the transcription factor is disabled by excision of the transcription factor by the recombinase. 
     
     
         7 . The transgenic non-human animal of  claim 1 , wherein expression of the transcription factor is enabled by expression of the recombinase. 
     
     
         8 . The transgenic non-human animal of  claim 7 , wherein the second heterologous nucleic acid comprises a transcriptional STOP signal preceding the transcription factor that prevents expression of the transcription factor, wherein the transcriptional STOP signal is flanked by recombinase recognition sites, such that expression of the transcription factor is enabled by excision of the transcriptional STOP signal by the recombinase. 
     
     
         9 . The transgenic non-human animal of  claim 1 , wherein the recombinase is a Cre recombinase, a flp recombinase or a β-recombinase. 
     
     
         10 . The transgenic non-human animal of  claim 1 , wherein the transcription factor is a tTA transcription factor or a fusion protein thereof. 
     
     
         11 . The transgenic non-human animal of  claim 1 , wherein the response element is a tetO transcription response element. 
     
     
         12 . The transgenic non-human animal of  claim 1 , wherein at least one of the first and second selective promoters comprises a tissue specific promoter. 
     
     
         13 . The transgenic non-human animal of  claim 1 , wherein at least one of the first and second selective promoters comprises a temporally specific promoter. 
     
     
         14 . The transgenic non-human animal of  claim 1 , wherein at least one of the first and second selective promoters comprises an inducible promoter. 
     
     
         15 . The transgenic non-human animal of  claim 1 , wherein at least one of the first and second selective promoters comprises a promoter that is selective for neural cells. 
     
     
         16 . An isolated transgenic cell from the transgenic non-human animal of  claim 1 . 
     
     
         17 . A method of producing a non-human animal that expresses a transgene in a selected population of cells, the method comprising:
 identifying at least one progeny of a cross between
 (a) a first transgenic non-human animal comprising first heterologous nucleic acid encoding a recombinase operably linked to a first selective promoter; and 
 (b) a second transgenic non-human animal comprising a second heterologous nucleic acid comprising a polynucleotide sequence that encodes a transcription factor operably linked to a second selective promoter, wherein the polynucleotide sequence is 
 (i) flanked by recombinase recognition sites; or 
 (ii) preceded by a transcription STOP signal flanked by recombinase recognition sites, 
   wherein the at least one identified progeny comprises the first and second heterologous nucleic acids and wherein the at least one progeny further comprises a transgene under the transcription regulatory control of a response element specific for the transcription factor.   
     
     
         18 . The method of  claim 17 , wherein the recombinase is expressed in a first population of cells and the transcription factor is expressed in a second population of cells, the first and second populations of cells comprising at least one overlapping sub-population of cells. 
     
     
         19 . The method of  claim 17 , wherein expression of the transcription factor is dependent on expression of the recombinase. 
     
     
         20 . The method of  claim 19 , wherein expression of the transcription factor is enabled by expression of the recombinase. 
     
     
         21 . The method of  claim 19 , wherein the expression of the transcription factor is disabled by expression of the recombinase. 
     
     
         22 . The method of  claim 17 , wherein at least one of the first and second selective promoters comprises a tissue specific promoter. 
     
     
         23 . The method of  claim 17 , wherein at least one of the first and second selective promoters comprises a temporally specific promoter. 
     
     
         24 . The method of  claim 17 , wherein at least one of the first and second selective promoters comprises an inducible promoter. 
     
     
         25 . The method of  claim 17 , wherein at least one of the first and second selective promoters comprises a promoter that is selective for neural cells. 
     
     
         26 . A method for selective expression of a transgene in a population of cells in an animal, comprising administering to the animal:
 a. a first viral vector comprising a nucleic acid encoding a recombinase operably linked to a nucleic acid encoding a first selective promoter;   b. a second viral vector comprising a nucleic acid encoding a transcription factor operably linked to a nucleic acid encoding a second selective promoter, wherein expression of the transcription factor is regulated by expression of the recombinase; and   c. a third viral vector comprising a transgene under the transcriptional control of a response element specific for the transcription factor.   
     
     
         27 . The method of  claim 26  wherein the first, second and third viral vectors are independently selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors and herpesvirus vectors. 
     
     
         28 . The method of  claim 26  wherein the transgene is a therapeutic molecule, a toxin or a maker protein. 
     
     
         29 . The method of  claim 28  wherein the therapeutic molecule is a wild-type gene or a pro-apoptotic molecule. 
     
     
         30 . The method of  claim 26  wherein the animal is a non-human animal. 
     
     
         31 . The method of  claim 26  wherein the animal is a human. 
     
     
         32 . A kit comprising:
 a. a first viral vector comprising a nucleic acid encoding a recombinase operably linked to a nucleic acid encoding a first selective promoter;   b. a second viral vector comprising a nucleic acid encoding a transcription factor operably linked to a nucleic acid encoding a second selective promoter; and   c. a third viral vector comprising a transgene under the transcriptional control of a response element specific for the transcription factor.   
     
     
         33 . The kit of  claim 32 , wherein the nucleic acid encoding the transcription factor is flanked by recognition sites for the recombinase. 
     
     
         34 . The kit of  claim 32 , wherein the second viral vector further comprises a transcription STOP signal flanked by recognition sites for the recombinase. 
     
     
         35 . A kit comprising:
 a. a first injection construct encoding a recombinase operably linked to a first selective promoter;   b. a second injection construct encoding a transcription factor operably linked to a second selective promoter; and   c. a third injection construct comprising a transgene under the transcriptional control of a response element specific for the transcription factor.   
     
     
         36 . The kit of  claim 35 , wherein the transcription factor is flanked by recognition sites for the recombinase. 
     
     
         37 . The kit of  claim 35 , wherein the second injection construct further comprises a transcription STOP signal flanked by recognition sites for the recombinase.